RESUMO
The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Ligação a DNA/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , Linhagem Celular , Proteínas de Ligação a DNA/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Sequências Repetidas Invertidas , Proteínas Recombinantes de Fusão/análise , Sequências Reguladoras de Ácido RibonucleicoRESUMO
The p53 tumor suppressor is a DNA-damage-responsive sequence-specific transcriptional activator. The sustained activation of the p53 response is incompatible with cell growth and viability. To circumvent this issue, a variety of negative feedback loops exist to limit the duration of p53 activation. Despite our understanding of p53 regulation, very little is known about the effect of transient p53 activation on the long-term expression of p53 target genes. Here we used a temperature-sensitive variant of p53 and oligonucleotide microarrays to monitor gene expression during and following reversible p53 activation. The expression of most p53-induced transcripts was rapidly reversible, consistent with active mRNA decay. Representative 3' UTRs derived from short-lived transcripts (i.e., DDB2 and GDF15) conferred instability on a heterologous mRNA, while 3' UTRs derived from more stable transcripts (i.e., CRYAB and TP53I3) did not. The 3' UTRs derived from unstable p53-induced mRNAs were significantly longer than those derived from stable mRNAs. These 3' UTRs had high uridine and low cytosine content, leading to a higher density of U-, AU-, and GU-rich sequences. Remarkably, short-lived p53 targets were induced faster, reaching maximum transcript levels earlier than the stable p53 targets. Taken together, the evidence indicates that the p53 transcriptional response has evolved with primarily short-lived target mRNAs and that post-transcription processes play a prominent role in the p53 response.
Assuntos
Regulação da Expressão Gênica , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regiões 3' não Traduzidas , Animais , Composição de Bases , Linhagem Celular Tumoral , Células HT29 , Humanos , Camundongos , Mutação/genética , Transcrição GênicaRESUMO
Background: Thapsigargin (Tg) is a compound that inhibits the SERCA calcium transporter leading to decreased endoplasmic reticulum (ER) Ca2+ levels. Many ER chaperones are required for proper folding of membrane-associated and secreted proteins, and they are Ca2+ dependent. Therefore, Tg leads to the accumulation of misfolded proteins in the ER, activating the unfolded protein response (UPR) to help restore homeostasis. Tg reportedly induces cell cycle arrest and apoptosis in many cell types but how these changes are linked to the UPR remains unclear. The activating transcription factor 4 (ATF4) plays a key role in regulating ER stress-induced gene expression so we sought to determine if ATF4 is required for Tg-induced cell cycle arrest and apoptosis using ATF4-deficient cells. Methods: Two-parameter flow cytometric analysis of DNA replication and DNA content was used to assess the effects of Tg on cell cycle distribution in isogenic HCT116-derived cell lines either expressing or lacking ATF4. For comparison, we similarly assessed the Tg response in isogenic cell lines deleted of the p53 tumour suppressor and the p53-regulated p21WAF1 cyclin-dependent kinase inhibitor important in G1 and G2 arrests induced by DNA damage. Results: Tg led to a large depletion of the S phase population with a prominent increase in the proportion of HCT116 cells in the G1 phase of the cell cycle. Importantly, this effect was largely independent of ATF4. We found that loss of p21WAF1 but not p53 permitted Tg treated cells to enter S phase and synthesize DNA. Therefore, p21WAF1plays an important role in these Tg-induced cell cycle alterations while ATF4 and p53 do not. Remarkably, the ATF4-, p53-and p21WAF1-deficient cell lines were all more sensitive to Tg-induced apoptosis. Taken together, p21WAF1 plays a larger role in regulating Tg-induced G1 and G2 arrests than ATF4 or p53 but these proteins similarly contribute to protection from Tg-induced apoptosis. This work highlights the complex network of stress responses that are activated in response to ER stress.
Assuntos
Fator 4 Ativador da Transcrição , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Tapsigargina/farmacologia , Fator 4 Ativador da Transcrição/genética , Linhagem Celular Tumoral , DNA , Quinases Ciclina-Dependentes/metabolismoRESUMO
Isoginkgetin (IGG) is a compound originally derived from the leaves of Ginkgo biloba trees. It was subsequently identified through a chemical screen to be an inhibitor of both the major and minor spliceosome, with an IC50 value of 30 µM [1]. Little is currently known about the overall effects of spliceosome inhibition on human cells. Here, we treated HCT116 and a p53 null subline of colon cancer cells with 30 µM IGG for 8 hours. Total RNA was isolated, and Affymetrix oligonucleotide microarray analysis was completed using samples from two biologically independent experiments. A relatively small number of transcripts were differentially expressed in these cell lines. There was considerable overlap in the upregulated but not the downregulated transcripts. PANTHER Reactome analysis of these shared upregulated transcripts identified enriched pathways involving the ATF4 transcription factor important in the integrated stress response [2].
RESUMO
RNA polymerase II is unable to bypass bulky DNA lesions induced by agents like ultraviolet light (UV light) and cisplatin that are located in the template strand of active genes. Arrested polymerases form a stable ternary complex at the site of DNA damage that is thought to pose an impediment to the repair of these lesions. Transcription-coupled nucleotide excision repair (TC-NER) preferentially repairs these DNA lesions through an incompletely defined mechanism. Based on elegant in vitro experiments, it was hypothesized that the transcription elongation factor IIS (TFIIS) may be required to couple transcription to repair by catalyzing the reverse translocation of the arrested polymerase, allowing access of repair proteins to the site of DNA damage. However the role of TFIIS in this repair process has not been tested in vivo. Here, silencing TFIIS using an RNA interference strategy did not affect the ability of cells to recover nascent RNA synthesis following UV exposure or the ability of cells to repair a UV-damaged reporter gene while a similar strategy to decrease the expression Cockayne syndrome group B protein (CSB) resulted in the expected repair defect. Furthermore, RNA interference against TFIIS did not increase the sensitivity of cells to UV light or cisplatin while decreased expression of CSB did. Taken together, these results indicate that TFIIS is not limiting for the repair of transcription-blocking DNA lesions and thus the present work does not support a role for TFIIS in TC-NER.
Assuntos
Reparo do DNA/genética , Interferência de RNA , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Células HCT116 , Humanos , Immunoblotting , Proteínas de Ligação a Poli-ADP-Ribose , Fatores de Elongação da Transcrição/metabolismo , Raios UltravioletaRESUMO
Isoginkgetin (IGG) is a small molecule inhibitor of pre-mRNA splicing. Failure to accurately remove introns could lead to the production of aberrant mRNAs and proteins. The cellular responses to splicing stress are not well defined. Here, we used oligonucleotide microarrays to assess genome wide changes in gene expression associated with exposure to IGG. Two of the 3 enriched pathways identified using PANTHER analysis of differentially expressed transcripts are linked to the ATF4 transcription factor. We confirmed that ATF4 was selectively translated and upregulated in response IGG despite an almost complete block to total protein synthesis. Importantly, partial disruption of the ATF4 gene using CRISPR-mediated gene editing prevented IGG-induced changes in gene expression. Remarkably, another spliceosome inhibitor, pladienolide B, did not inhibit translation, activate ATF4 or increase ATF4-dependent gene expression. Taken together, IGG activates ATF4 and an ATF4-dependent transcriptional response but these effects are not common to all spliceosome inhibitors.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Biflavonoides/farmacologia , Fator 2 Ativador da Transcrição/genética , Células HeLa , Células Hep G2 , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. METHODS: RNA interference (RNAi) was used to reduce the transcription-coupled nucleotide excision repair (TC-NER) capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B) transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. RESULTS: These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. CONCLUSION: The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Colorretais/patologia , Reparo do DNA , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos da radiação , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Proteínas de Ligação a Poli-ADP-Ribose , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Interferência de RNA , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta , Regulação para Cima , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
The MDM2 oncogene is a negative regulator of the p53 tumour suppressor. This relationship appears to have originated over a billion years ago. The human MDM2 gene encodes a variety of mRNAs with exceptionally long 3'UTRs (up to 5.7 kb); however, it was unclear whether MDM2 3'UTRs from other species are similarly long or conserved at the sequence level. Here, we report that all but one of the primate species most closely related to humans (greater and lesser apes) have similarly long 3'UTRs with high sequence similarity across their entire length. More distantly related species (Old world monkeys and new world monkeys) tend to have shorter MDM2 3'UTRs homologous to the corresponding position of the human MDM2 3'UTR while non-primate species exhibit little similarity at all. Remarkably, DNA sequences downstream of the shorter primate 3'UTRs are syntenic with distal regions in the human and other ape MDM2 3'UTRs. These homologous non-transcribed intergenic and transcribed 3'UTR-encoding regions are comprised of a variety of transposable elements, an RLP24 pseudogene and a cluster of novel repeat sequences suggestive of another unknown transposable element. Our analysis suggests that the primary difference between long and short MDM2 3'UTRs is a switch in polyA site usage to include conserved transposable elements that remain intergenic in more distantly related primates. It will be important to determine the relative contribution of these elements to post-transcriptional and translational regulation of MDM2 and hence p53-mediated tumour suppression.
Assuntos
Evolução Molecular , Genômica , Primatas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Regiões 3' não Traduzidas/genética , Animais , Hibridização Genômica Comparativa , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Poliadenilação/genética , Pseudogenes/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Biflavonoides/farmacologia , Morte Celular/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Fibroblastos/efeitos dos fármacos , Macrolídeos/farmacologia , Spliceossomos/metabolismo , Fator 3 Ativador da Transcrição/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Morte Celular/fisiologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Camundongos , RNA Interferente PequenoRESUMO
The spliceosome is a large ribonucleoprotein complex that catalyzes the removal of introns from RNA polymerase II-transcribed RNAs. Spliceosome assembly occurs in a stepwise manner through specific intermediates referred to as pre-spliceosome complexes E, A, B, B* and C. It has been reported that small molecule inhibitors of the spliceosome that target the SF3B1 protein component of complex A lead to the accumulation of cells in the G1 and G2/M phases of the cell cycle. Here we performed a comprehensive flow cytometry analysis of the effects of isoginkgetin (IGG), a natural compound that interferes with spliceosome assembly at a later step, complex B formation. We found that IGG slowed cell cycle progression in multiple phases of the cell cycle (G1, S and G2) but not M phase. This pattern was somewhat similar to but distinguishable from changes associated with an SF3B1 inhibitor, pladienolide B (PB). Both drugs led to a significant decrease in nascent DNA synthesis in S phase, indicative of an S phase arrest. However, IGG led to a much more prominent S phase arrest than PB while PB exhibited a more pronounced G1 arrest that decreased the proportion of cells in S phase as well. We also found that both drugs led to a comparable decrease in the proportion of cells in M phase. This work indicates that spliceosome inhibitors affect multiple phases of the cell cycle and that some of these effects vary in an agent-specific manner despite the fact that they target splicing at similar stages of spliceosome assembly.
Assuntos
Biflavonoides/farmacologia , Divisão Celular/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Fase S/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Citometria de Fluxo , Células HCT116 , Humanos , Macrolídeos/farmacologia , Precursores de RNA/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismoRESUMO
Mammaglobin B (MGB2) and mammaglobin A (MGB1) are proteins expressed in metastatic breast cancers. The early detection of circulating tumor cells (CTCs) in breast cancer patients is crucial to decrease mortality rate. Herein, novel aptamers were successfully selected and characterized against MGB2 and MGB1 proteins using a hybrid SELEX approach. The potential use of the selected aptamers in breast CTC detection was studied using spiked breast cancer cells in whole blood lysate. The results obtained from this study showed that the selected aptamers (MAMB1 and MAMA2) bind to their target breast cancer cell lines with high affinity (low nanomolar Kd values) and specificity. They also bind to their free recombinant target proteins and show minimal non-specific binding to normal and other cancer cell lines. Additionally, they were able to distinguish a low number of breast cancer cells spiked in whole blood lysate containing normal blood cells. The results obtained in this study indicate the great potential for the use of aptamers to detect MGB1 and MGB2 protein biomarkers, expressed on the surface of breast CTCs.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias da Mama/sangue , Mamoglobina A/sangue , Mamoglobina B/sangue , Células Neoplásicas Circulantes/metabolismo , Técnica de Seleção de Aptâmeros , Biomarcadores Tumorais/sangue , Linhagem Celular , Biologia Computacional , Citometria de Fluxo , Testes Hematológicos/métodos , Humanos , Microscopia de Fluorescência , Técnica de Seleção de Aptâmeros/métodos , Sensibilidade e EspecificidadeRESUMO
The p53 tumour suppressor is a transcription factor that can increase the expression of mRNAs and microRNAs (miRNAs). HT29-tsp53 cells expressing a temperature sensitive variant of p53 have provided a useful model to rapidly and reversibly control p53 activity. In this model, the majority of p53-responsive mRNAs were upregulated rapidly but they were short-lived leading to rapid decay of the p53 response at the restrictive temperature. Here we used oligonucleotide microarrays and reverse transcriptase PCR to show that p53-induced miRNAs exhibited a distinct temporal pattern of expression. Whereas p53-induced miRNAs like miR-143-3p, miR-145-5p, miR-34a-5p and miR-139-5p increased as fast as mRNAs, they were extremely stable persisting long after p53 induced mRNAs and even their corresponding primary miRNAs had decayed to baseline levels. Three p53-induced mRNAs (MDM2, BTG2 and CDKN1A) are experimentally verified targets of one or more of these specific miRNAs so we hypothesized that the sustained expression of p53-induced miRNAs could be explained by a post-transcriptional feedback loop. Activation of consecutive p53 responses separated by a period of recovery led to the selective attenuation of a subset of p53 regulated mRNAs corresponding to those targeted by one or more of the p53-responsive miRNAs. Our results indicate that the long term expression of p53 responsive miRNAs leads to an excess of miRNAs during the second response and this likely prevents the induction of MDM2, BTG2 and CDKN1A mRNA and/or protein. These observations are likely to have important implications for daily cancer therapies that activate p53 in normal tissues and/or tumour cells.
Assuntos
MicroRNAs/genética , Estabilidade de RNA , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/fisiologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , MicroRNAs/fisiologia , Análise em Microsséries , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Mensageiro/metabolismo , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs.
Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , MicroRNAs/biossíntese , RNA Neoplásico/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , MicroRNAs/genética , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Lack of functional pRb results in attenuated recovery of mRNA synthesis and increased apoptosis following UV radiation in human breast cancer cells. We have previously demonstrated that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (pRb), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15-20-fold more sensitive to UV radiation than cells with wild-type pRb. In order to understand the mechanisms of the high apoptotic response of MDA-MB-468 cells to UV radiation, we examined the effects of UV on these cells with regards to both membrane-mediated events and DNA damage. We found that MDA-MB-468 cells were resistant to all ligand-induced death receptor signaling. In addition, although UV activated caspase 8 in MDA-MB-468 cells, a peptide inhibitor of caspase 8 failed to inhibit UV-induced apoptosis. We then tested the possibility that nuclear events mediated the enhanced sensitivity to UV-induced apoptosis in these cells. Unlike UV-resistant cells, MDA-MB-468 cells were unable to recover mRNA synthesis after 5 J/m2 UVC. We also found that the pRb-null DU-145 cells similarly had attenuated recovery of mRNA synthesis after UV radiation. In UV-resistant cells with wild-type pRb, the inactivation of pRb with HPV-16 E7 resulted in significant inhibition in their ability to recover mRNA synthesis and increased levels of apoptosis following UV radiation. Furthermore, pRb-null cells were deficient in repair of UV radiation-induced DNA damage. These data suggest that the sensitivity of MDA-MB-468 cells to UV radiation is due to defects in repair of DNA damage and recovery of mRNA synthesis rather than to membrane death receptor pathways. Inactivation of pRb may contribute to an increased sensitivity to UV radiation by attenuating repair of DNA lesions and recovery of mRNA synthesis following UV radiation.
Assuntos
Apoptose/efeitos da radiação , RNA Mensageiro/biossíntese , Proteína do Retinoblastoma/metabolismo , Raios Ultravioleta/efeitos adversos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspase 8 , Caspase 9 , Caspases/metabolismo , Dano ao DNA/efeitos da radiação , Reparo do DNA/fisiologia , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/genética , Células Tumorais CultivadasRESUMO
The transcription-coupled nucleotide excision repair (TCNER) pathway maintains genomic stability by rapidly eliminating helix-distorting DNA adducts, such as UV-induced cyclobutane pyrimidine dimers (CPDs), specifically from the transcribed strands of active genes. DNA mismatch repair (MMR) constitutes yet another critical antimutagenic pathway that removes mispaired bases generated during semiconservative replication. It was previously reported that the human colon adenocarcinoma strains HCT116 and LoVo (bearing homozygous mutations in the MMR genes hMLH1 and hMSH2, respectively), besides manifesting hallmark phenotypes associated with defective DNA mismatch correction, are also completely deficient in TCNER of UV-induced CPDs. This revealed a direct mechanistic link between MMR and TCNER in human cells, although subsequent studies have either supported, or argued against, the validity of this important notion. Here, the ligation-mediated polymerase chain reaction was used to show at nucleotide resolution that MMR-deficient HCT116 and LoVo retain the ability to excise UV-induced CPDs much more rapidly from the transcribed vs the nontranscribed strands of active genes. Moreover, relative to DNA repair-proficient counterparts, MMR-deficient cells were not more sensitive to the cytotoxic effects of UV, and displayed equal ability to recover mRNA synthesis following UV challenge. These results conclusively demonstrate that hMLH1- and hMSH2-deficient human colon adenocarcinoma cells are fully proficient in TCNER.
Assuntos
Pareamento Incorreto de Bases , Reparo do DNA/genética , Proteínas de Ligação a DNA , Mutação , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Humanos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Reação em Cadeia da Polimerase , Dímeros de Pirimidina/metabolismo , Transcrição GênicaRESUMO
Transcription-coupled nucleotide excision repair (tcNER)-deficient human fibroblasts are extremely sensitive to the induction of apoptosis in response to low doses of ultraviolet light (UV light), but are less sensitive to the induction of apoptosis following exposure to high doses [J. Invest. Dermatol. 117 (2001) 1162]. This seemingly paradoxical observation led us to re-evaluate the relationship between UV dose and the induction of apoptosis. Here we report that the reduction in the extent of UV-induced apoptosis in tcNER-deficient strains following exposure to elevated doses of UV light does not result from impaired gene expression alone because neither inhibitors of transcription nor inhibitors of translation blocked UV-induced apoptosis. Furthermore, UV-induced apoptosis was greatly reduced by inhibiting S-phase progression with either mimosine or serum withdrawal. Importantly, DNA synthesis following UV-irradiation occurred only at doses that induced apoptosis in these cell lines and the apoptotic cells contained nascent DNA. Moreover, deregulation of G(1)- to S-phase transition by expression of human papillomavirus E7 sensitized cells to UV-induced apoptosis. Taken together these results suggest that the induction of apoptosis requires S-phase progression following UV-irradiation.
Assuntos
Apoptose/efeitos da radiação , Fase S/fisiologia , DNA Helicases , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Fase G1/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Mimosina/farmacologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
SIGNIFICANCE: Production of proteins requires the synthesis, maturation, and export of mRNAs before their translation in the cytoplasm. Endogenous and exogenous sources of DNA damage pose a challenge to the co-ordinated regulation of gene expression, because the integrity of the DNA template can be compromised by DNA lesions. Cells recognize and respond to this DNA damage through a variety of DNA damage responses (DDRs). Failure to deal with DNA damage appropriately can lead to genomic instability and cancer. RECENT ADVANCES: The p53 tumor suppressor plays a dominant role in DDR-dependent changes in gene expression, but this transcription factor is not solely responsible for all changes. Recent evidence indicates that RNA metabolism is integral to DDRs as well. In particular, post-transcriptional processes are emerging as important contributors to these complex responses. CRITICAL ISSUES: Transcriptional, post-transcriptional, and translational regulation of gene expression is subject to changes in response to DNA damage. How these processes are intertwined in the unfolding of DDR is not fully understood. FUTURE DIRECTIONS: Many complex regulatory responses combine to determine cell fate after DNA damage. Understanding how transcriptional, post-transcriptional, and translational processes interdigitate to create a web of regulatory interactions will be one of the key challenges to fully understand DDRs.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA , Interferência de RNA , Animais , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Estabilidade de RNA , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , Raios UltravioletaRESUMO
Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER(+)) breast cancers. There are two ER subtypes, ERα and ERß, which promote and inhibit breast cancer cell proliferation, respectively. Although ER(+) breast cancers typically express a high ratio of ERα to ERß, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERß. On some gene enhancers, ERß has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERß agonists, WAY-20070 and a novel "A-CD" estrogen called L17, produce a marked reduction in G(2)-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERß agonists recruited both the ERα and ERß to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERß-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERß. Exposure to the ERß ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERß agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERß agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Oxazóis/farmacologia , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Ligantes , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Bulky DNA adducts induced by agents like ultraviolet light, cisplatin and oxidative metabolism pose a block to elongation by RNA polymerase II (RNAPII). The arrested RNAPII can initiate the repair of transcription-blocking DNA lesions by transcription-coupled nucleotide excision repair (TC-NER) to permit efficient recovery of mRNA synthesis while widespread sustained transcription blocks lead to apoptosis. Therefore, RNAPII serves as a processive DNA damage sensor that identifies transcription-blocking DNA lesions. Cockayne syndrome (CS) is an autosomal recessive disorder characterized by a complex phenotype that includes clinical photosensitivity, progressive neurological degeneration and premature-aging. CS is associated with defects in TC-NER and the recovery of mRNA synthesis, making CS cells exquisitely sensitive to a variety of DNA damaging agents. These defects in the coupling of repair and transcription appear to underlie some of the complex clinical features of CS. Recent insight into the consequences of blocked transcription and their relationship to CS will be discussed.
Assuntos
Síndrome de Cockayne/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Transcrição Gênica , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , Adutos de DNA/genética , Humanos , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , RNA Polimerase II/genética , RNA Mensageiro/genética , Raios Ultravioleta/efeitos adversosRESUMO
The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1ß apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1ß expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.